Normal muscle fiber type distribution is recapitulated in aged ephrin-A3-/- mice that previously lacked most slow myofibers.

Individual limb muscles have characteristic representation and spatial distribution of muscle fiber types (one slow and up to three fast isoforms) appropriate to their unique anatomical location and function. This distribution can be altered by physiological stimuli such as training (i.e., for increased endurance or force) or pathological conditions such as aging. Our group previously showed that ephrin-A3 is expressed only on slow myofibers, and that adult mice lacking ephrin-A3 have dramatically reduced numbers of slow myofibers due to postnatal innervation of previously slow myofibers by fast motor neurons. In this study, fiber type composition of hindlimb muscles of aged and denervated/reinnervated C57BL/6 and ephrin-A3-/- mice was analyzed to determine whether the loss of slow myofibers persists across the lifespan. Surprisingly, fiber-type composition of ephrin-A3-/- mouse muscles at two years of age was nearly indistinguishable from age-matched C57BL/6 mice. After challenge with nerve crush, the percentage of IIa and I/IIa hybrid myofibers increased significantly in aged ephrin-A3-/- mice. While EphA8, the receptor for ephrin-A3, is present at all neuromuscular junctions (NMJs) on fast fibers in 3-6 mo old C57BL/6 and ephrin-A3-/- mice, this exclusive localization is lost with aging, with EphA8 expression now found on a subset of NMJs on some slow muscle fibers. This return to appropriate fiber-type distribution given time and under use reinforces the role of activity in determining fiber-type representation and suggests that, rather than being a passive baseline, the developmentally and evolutionarily selected fiber type pattern may instead be actively reinforced by daily living.

EphA7 promotes myogenic differentiation via cell-cell contact.

The conversion of proliferating skeletal muscle precursors (myoblasts) to terminally-differentiated myocytes is a critical step in skeletal muscle development and repair. We show that EphA7, a juxtacrine signaling receptor, is expressed on myocytes during embryonic and fetal myogenesis and on nascent myofibers during muscle regeneration in vivo. In EphA7-/- mice, hindlimb muscles possess fewer myofibers at birth, and those myofibers are reduced in size and have fewer myonuclei and reduced overall numbers of precursor cells throughout postnatal life. Adult EphA7-/- mice have reduced numbers of satellite cells and exhibit delayed and protracted muscle regeneration, and satellite cell-derived myogenic cells from EphA7-/- mice are delayed in their expression of differentiation markers in vitro. Exogenous EphA7 extracellular domain will rescue the null phenotype in vitro, and will also enhance commitment to differentiation in WT cells. We propose a model in which EphA7 expression on differentiated myocytes promotes commitment of adjacent myoblasts to terminal differentiation.

Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging.

Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain.

Ephrin-A3 promotes and maintains slow muscle fiber identity during postnatal development and reinnervation.

Each adult mammalian skeletal muscle has a unique complement of fast and slow myofibers, reflecting patterns established during development and reinforced via their innervation by fast and slow motor neurons. Existing data support a model of postnatal "matching" whereby predetermined myofiber type identity promotes pruning of inappropriate motor axons, but no molecular mechanism has yet been identified. We present evidence that fiber type-specific repulsive interactions inhibit innervation of slow myofibers by fast motor axons during both postnatal maturation of the neuromuscular junction and myofiber reinnervation after injury. The repulsive guidance ligand ephrin-A3 is expressed only on slow myofibers, whereas its candidate receptor, EphA8, localizes exclusively to fast motor endplates. Adult mice lacking ephrin-A3 have dramatically fewer slow myofibers in fast and mixed muscles, and misexpression of ephrin-A3 on fast myofibers followed by denervation/reinnervation promotes their respecification to a slow phenotype. We therefore conclude that Eph/ephrin interactions guide the fiber type specificity of neuromuscular interactions during development and adult life.

Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength.

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.

Eph/ephrin interactions modulate muscle satellite cell motility and patterning.

During development and regeneration, directed migration of cells, including neural crest cells, endothelial cells, axonal growth cones and many types of adult stem cells, to specific areas distant from their origin is necessary for their function. We have recently shown that adult skeletal muscle stem cells (satellite cells), once activated by isolation or injury, are a highly motile population with the potential to respond to multiple guidance cues, based on their expression of classical guidance receptors. We show here that, in vivo, differentiated and regenerating myofibers dynamically express a subset of ephrin guidance ligands, as well as Eph receptors. This expression has previously only been examined in the context of muscle-nerve interactions; however, we propose that it might also play a role in satellite cell-mediated muscle repair. Therefore, we investigated whether Eph-ephrin signaling would produce changes in satellite cell directional motility. Using a classical ephrin 'stripe' assay, we found that satellite cells respond to a subset of ephrins with repulsive behavior in vitro; patterning of differentiating myotubes is also parallel to ephrin stripes. This behavior can be replicated in a heterologous in vivo system, the hindbrain of the developing quail, in which neural crest cells are directed in streams to the branchial arches and to the forelimb of the developing quail, where presumptive limb myoblasts emigrate from the somite. We hypothesize that guidance signaling might impact multiple steps in muscle regeneration, including escape from the niche, directed migration to sites of injury, cell-cell interactions among satellite cell progeny, and differentiation and patterning of regenerated muscle.

Neural crest cell communication involves an exchange of cytoplasmic material through cellular bridges revealed by photoconversion of KikGR.

Neural crest (NC) cells invade the vertebrate embryo in ordered migratory streams, yet it is unclear whether cells communicate to maintain spacing and direction. Here, we examined NC cell communication in detail, using optical highlighting and photobleaching to monitor cell contact dynamics. We observed cytoplasmic transfer between NC cell neighbors through thin cellular bridges. The transfer of molecules between NC cells was bi-directional, not at equal rates, and independent of bridge dynamics. The cytoplasmic transfer was prevalent in recently divided NC cells. Molecular simulations, based on Brownian motion and measured cell volumes, predicted that simple diffusion could not account for observed cytoplasmic transfer rates. Cell tracking revealed that exchange of cytoplasmic material preceded the re-orientation of cells to the direction of migration. Our data suggest a mechanism by which NC cells communicate position information through the formation of cellular bridges that allow exchange of cytoplasmic material through active transport.

Watching the assembly of an organ a single cell at a time using confocal multi-position photoactivation and multi-time acquisition.

Tracing cell movements in vivo yields important clues to organogenesis, yet it has been challenging to accurately and reproducibly fluorescently mark single and small groups of cells to build a picture of tissue assembly. In the early embryo, the small size (hundreds of cells) of progenitor cell regions has made it easier to identify and selectively mark superficially located cells by glass needle injection. However,during early organogenesis,subregions of interest may be several millions of cells in volume located deeper within the embryo requiring an alternative approach. Here, we combined (confocal and 2-photon) photoactivation cell labeling and multi-position, multi-time imaging to trace single cell and small subgroups of cells in the developing brain and spinal cord. We compared the photostability and photoefficiency of a photoswitchable fluorescent protein, PSCFP2, with a novel nuclear localized H2B-PSCFP2 protein. We showed that both fluorescent proteins have similar photophysical properties and H2B-PSCFP2 is more effective in single cell identification in dense tissue. To accurately and reproducibly fluorescently trace subregions of cells in a 3D tissue volume, we developed a protocol for multi-position photoactivation and multi-time acquisition in the chick spinal cord in up to eight tissue sections. We applied our techniques to address the formation of the sympathetic ganglia,a major component of the autonomic nervous system,and showed there are phenotypic differences between early and later emerging neural crest cells and their positions in the developing ganglia. Thus, targeted fluorescent cell marking by confocal or 2-photon multi-position photoactivation and multi-time acquisition offer a more efficient, less invasive technique to trace cell movements in large regions of interest and move us closer towards mapping the cellular events of organogenesis.

Neural crest invasion is a spatially-ordered progression into the head with higher cell proliferation at the migratory front as revealed by the photoactivatable protein, KikGR.

Neural crest cell (NCC) invasion is a complex sculpting of individual cells into organized migratory streams that lead to organ development along the vertebrate axis. Key to our understanding of how molecular mechanisms modulate the NCC migratory pattern is information about cell behaviors, yet it has been challenging to selectively mark and analyze migratory NCCs in a living embryo. Here, we apply an innovative in vivo strategy to investigate chick NCC behaviors within the rhombomere 4 (r4) migratory stream by combining photoactivation of KikGR and confocal time-lapse analysis of H2B-mRFP1 transfected NCCs. We find that the spatial order of r4 NCC emergence translates into a distal-to-proximal invasion of the 2nd branchial arch. Lead and trailing NCCs display similar average cell speeds and directionalities. Surprisingly, we find that lead NCCs proliferate along the migratory route and grow to outnumber trailing NCCs by nearly 3 to 1. A simple, cell-based computational model reproduces the r4 NCC migratory pattern and predicts the invasion order can be disrupted by slower, less directional lead cells or by environmental noise. Our results suggest a model in which NCC behaviors maintain a spatially-ordered invasion of the branchial arches with differences in cell proliferation between the migratory front and trailing NCCs.

Photoactivation cell labeling for cell tracing in avian development.

INTRODUCTIONTracing cell movements in a living embryo or embryo slice culture remains a challenging problem due to difficulties in cell accessibility and in accurate delivery of fluorescent labels into an individual cell or subgroup of cells. Here, we describe a photoactivation cell-labeling technique in avian embryos that allows for selective marking of individual cells or groups of cells at precise times and spatial locations normally not accessible using previous techniques. The current protocol is also less invasive than previously described methods. We provide details of fluorescent protein delivery into cells of interest utilizing microinjection and in ovo electroporation, as well as the optical parameters needed for photoactivation and imaging of both single- and dual-color photoactivatable fluorescent proteins (PAFPs). We present applications to label single cells and small subgroups of cells throughout the head and trunk of the developing vertebrate embryo, using the avian neural crest as our model cell population.

An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model.

Tracing the lineage or neighbor relationships of cells in a migratory population or deep within an embryo is difficult with current methods. The recent explosion of photoactivatable fluorescent proteins (PAFPs) offers a unique cell labeling tool kit, yet their in vivo performance in intact embryos and applicability have not been thoroughly explored. We report a comparison study of PAGFP, PSCFP2, KikGR, and Kaede analyzed in the avian embryo using confocal and 2-photon microscopy. PAFPs were introduced into the chick neural tube by electroporation and each photoconverted in the neural crest or cells in the neural tube with exposure to 405 nm light, but showed dramatic differences in photoefficiency and photostability when compared at the same 2% laser power. KikGR and Kaede photoconverted with ratios only slightly lower than in vitro results, but cells rapidly photobleached after reaching maximal photoefficiency. PSCFP2 had the lowest photoefficiency and photoconverted nearly 70 times slower than the other dual-color PAFPs tested, but was effective at single-cell marking, especially with 2-photon excitation at 760 nm. The dual-color PAFPs were more effective to monitor cell migratory behaviors, since non-photoconverted neighboring cells were fluorescently marked with a separate color. However, photoconverted cells were limited in all cases to be visually distinguishable for long periods, with PSCFP2 visible from background the longest (48 hr). Thus, photoactivation in embryos has the potential to selectively mark less accessible cells with laser accuracy and may provide an effective means to study cell-cell interactions and short-term cell lineage in developmental and stem cell biology.

Photoactivatable green fluorescent protein as a single-cell marker in living embryos.

Selective marking of a single cell within an embryo is often difficult to perform with existing methods. Here, we report a minimally invasive optical technique that uses 405-nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). Single cells and small groups of cells (n < 10) are successfully marked, from a region of cells injected and electroporated with PAGFP, in both whole chick embryo explants and in ovo. Photoactivated cells display normal cell migratory behaviors and retain a bright GFP signal for at least 24 hr when followed with confocal time-lapse microscopy. We determined that using a low-magnification objective (approximately x 10) and low laser power (approximately 1-10%) leads to a steady increase in fluorescence signal within a photoactivated cell and minimizes photobleaching. The utility of PAGFP photoactivation was tested to address a specific question in developmental biology. Specifically, we asked whether neighboring migratory cells that emerge from the hindbrain and invade surrounding peripheral tissues maintain neighbor relationships while traveling to the destination sites. We found that some neural crest do not maintain neighbor relationships, such that two neighboring cells near the neural tube cells may populate different branchial arches. The ability to optically photoactivate PAGFP in a single or small group of cells and follow individual cell migratory behaviors within a living embryo offers a powerful, minimally invasive cell marking tool for precise, in vivo cell migration studies.